Embryonic mouse spinal neurons and clonal rat pituitary cells have been studied with a variety of techniques current in cellular neurobiology, including dissociated cell culture, intact-cell ligand binding, immunohistochemistry, in vitro immunization, hybridoma cloning, immunoblot analysis, retrograde transport of fluorescent labe, flow cytometry, and electron microscopy. The initial focus of this project is to develop a successful multi-disciplinary strategy aimed at studying the differentiation of specific cellular properties important in neuronal function. Protocols for maintaining dissociated cultures of primary neurons and clonal pituitary cells have been improved. Binding assays to intact central neurons under conditions identical to those used in performing electrophysiological recordings have been performed. New techniques for generating primary immune responses rapidly in vitro have been successfully developed and with these techniques a panel of monoclonal antibodies has been produced. Initial screening shows that two antibodies complex with surface antigens expressed both in cultured clonal pituitary cells and in spinal cord neurons in vivo and in vitro. Accummulation of retrograde transported fluorescent dye by embryonic motoneurons has been used to provide a signal for fluorescent-activated cell sorting. Flow cytometry has also been used in conjunction with fluorescent immunohistochemical reactions of cells in suspension to provide a quantitative analysis of the development of specific populations of spinal cord cells. Flow cytometric isolation of fluorescently labeled cells should yield relatively pure populations of specific cell types both for use as complex antigen in generating specific immunoreagents and for multidisciplinary analysis. With this multi-disciplinary strategy we should be able to study specific types of cells at relatively quantitative levels of analysis and gain some insight into their biological properties, as well as how these properties come to be expressed during embryogenesis and what roles they play in the synthetic and secretory functions asociated with the different types.